Fusion immunomodulatory proteins and methods for making same

ABSTRACT

The present invention relates generally to the field of generating recombinant chimeric fusion proteins to be used in the cancer therapy, and more specifically, to fusion molecules of Anti-EGFR1-TGFβRII, Anti-EGFR1-PD1 and Anti-CTLA4-PD1 and methods of generating same, wherein the methods reduce production costs and increase homogeneity of the recombinant chimeric fusion proteins.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application and claims priority tocopending U.S. patent application Ser. No. 15/968,122 filed on May 1,2018 and now U.S. Pat. No. 10,766,963, which in turn is a divisional andclaims priority to copending U.S. patent application Ser. No. 14/771,308filed on Aug. 28, 2015 and now U.S. Pat. No. 9,988,456 which was filedunder the provisions of 35 U.S.C. § 371 and claims the priority ofInternational Patent Application No. PCT/US2014/022404 filed on Mar. 10,2014 which in turn claims priority to U.S. Provisional PatentApplication No. 61/777,016 filed on Mar. 12, 2013 the contents of whichare hereby incorporated by reference herein for all purposes.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates generally to the field of generatingrecombinant chimeric fusion proteins to be used in the cancer therapy,and more specifically, to fusion molecules of Anti-EGFR1-TGFβRII,Anti-EGFR1-PD1 and Anti-CTLA4-PD1 and methods of generating same,wherein the methods reduce production costs and increase homogeneity ofthe recombinant chimeric fusion proteins.

Related Art

In spite of numerous advances in medical research, cancer remains thesecond leading cause of death in the United States. Traditional modes ofclinical care, such as surgical resection, radiotherapy andchemotherapy, have a significant failure rate, especially for solidtumors. Failure occurs either because the initial tumor is unresponsive,or because of recurrence due to regrowth at the original site ormetastasis. Cancer remains a central focus for medical research anddevelopment.

Immunotherapy of cancer has been explored for over a century, but it isonly in the last decade that various antibody-based products have beenintroduced into the management of patients with diverse forms of cancer.At present, this is one of the most active areas of clinical research,with numerous antibody therapeutic products already approved inoncology.

Using specific antibodies as therapeutic agents offers advantages overthe non-targeted therapies such as systemic chemotherapy via oral orintravenous administration of drugs or radiation therapy. There are twotypes of antibody-based therapies. The more common type is to identify atumor antigen (i.e., a protein expressed on tumors and cancer cells andnot in normal tissues) and develop an antibody, preferably a monoclonalantibody (mAb) directed to the tumor antigen. One can then conjugate anytherapeutic agent, such as a chemotherapeutic agent, a radionuclide,modified toxin, etc., to this antibody to achieve targeted therapy bythe therapeutic agent to the tumor. The other kind of antibody basedtherapy is by providing an antibody which in itself has therapeuticproperties against the tumor/cancer cells it targets. The addedadvantage of this second form of antibody-based therapy is that one mayadditionally conjugate another therapeutic agent to the therapeuticantibody to achieve a more effective treatment. The major advantage withany antibody-directed therapy and of therapy using monoclonal antibodies(mAbs) in particular, is the ability to deliver increased doses of atherapeutic agent to a tumor, with greater sparing of normal tissue fromthe side effects of the therapeutic agent.

Despite the identification of several antibodies for cancer therapies,there is still a need to identify new and more effective therapeutics toovercome immune tolerance and activate T cell responses. Further, eventhough molecular engineering has improved the prospects for suchantibody-based therapeutics issues still remain regarding continuity inthe generated recombinant products.

SUMMARY OF THE INVENTION

The present invention provides for a novel and consistent synthesismethod for generating homogeneous recombinant fusion immunomodulatorymolecules, and more specifically, recombinant chimeric polypeptidesincluding targeting antibodies linked to immunomodulatory proteins.

To mediate an immune response against cancer, T cell activation andco-stimulation are both important. Co-stimulation of T cells is mainlymediated through engaging of CD28 with its ligands of B7 family onantigen presenting cells (APCs). However, after activation, T cellsexpress a molecule called Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4),which binds to B7 ligands with much more affinity than CD28 and suchbinding down-modulates T cell activity. Thus, including an antibody thatbinds to the CTLA-4 receptor would block interaction with ligands of theB7 family and enhance anti-tumor response.

Programmed Death Ligand-1 (PDL1), one of the B7 ligands discussed above,obstructs anti-tumor immunity by (i) tolerizing tumor-reactive T cellsby binding to its receptor PD1 (CD279) on T cells; (ii) rendering tumorcells resistant to CD8+ T cell and FasL-mediated lysis by PD-1 signalingthrough tumor cell-expressed PDL1; and (iii) promoting the developmentand maintenance of induced T regulatory cells. Therefore, PDL1 is amajor obstacle to natural anti-tumor immunity and to cancerimmunotherapies requiring activation of host T cell-mediated anti-tumorimmunity. This concept is supported by studies demonstrating thatantibody blocking of PDL1-PD1 interactions improves T cell activationand reduces tumor progression. Although antibodies to PDL1 or PD1 haveshown therapeutic efficacy in a subset of cancer patients, the majorityof patients do not benefit from antibody treatment. Thus, there isneeded a mechanism for regulating PD-L1 function that will lead to a newuniversally applicable treatment for minimizing PD-L1-mediated immunesuppression in cancer patients and that is more effective than currentlyavailable mAbs to PD-1 or PD-L1.

A characteristic of many epithelial cancers, such as, cancers of thecolon, head and neck, breast, ovary, non-small cell lung (NSCL), andpancreas, is abnormally high levels of epidermal growth factor receptor(EGFR) on the surface of cancer cells. The family of epidermal growthfactor receptors (EGFR; HER1, HER2/neu, HER3, and HER4) includes cellmembrane receptors with intrinsic tyrosine kinase activity that triggera cascade of biophysiological signaling reactions in response to thebinding of different ligands. These receptors play a key role in thebehavior of malignant cells in a variety of human tumors, inducingincreased proliferation, decreasing apoptosis, and enhancing tumor cellmotility and angiogenesis. Thus, the present invention includesantibodies targeting EGFR family members.

The present invention further provides methods of reducing growth ofcancer cells by counteracting immune tolerance of cancer cells, whereinT cells remain active and inhibit the recruitment of T-regulatory thatare known to suppress the immune system's response to the tumor. Thus,the chimeric polypeptides generated by the polynucleotides sequences ofthe present invention are useful for treating cancer because of theexpressed fusion or chimeric polypeptides.

In one aspect, the present invention provides for chimeric polypeptidescontaining at least one targeting moiety to target a cancer cell and atleast one immunomodulating moiety that counteracts immune tolerance ofcancer cell, wherein the targeting moiety and the immunomodulatingmoiety are linked by an amino acid spacer of sufficient length of aminoacid residues so that both moieties can successfully bond to theirindividual target. In the alternative, the targeting moiety and theimmunomodulating moiety that counteract immune tolerance of cancer cellmay be bound directly to each other. The chimeric/fusion polypeptides ofthe invention are useful for binding to a cancer cell receptor andreducing the ability of cancer cells to avoid an immune response.

Preferably the targeting moiety is an antibody having binding affinityfor CTLA-4 or EGFR1, wherein the antibody is transcribed from apolynucleotide sequence lacking nucleotides for expression of theC-terminal lysine of the heavy chain of the expressed antibody. It hasbeen discovered that by removing the C-terminal lysine of the heavychain of an antibody during transcription that the end product exhibitsincreased homogeneity, thereby reducing the need and costs for furtherpurification.

It is known that during the process of transcription and translation ofan IgG molecule in CHO cells, the lysine (K) at the C-terminal of theheavy chain will be expressed. In the commercial product such expressedlysines have to be removed to increase purity. There is muchheterogeneity in the produced product, as shown in FIG. 1. This occursbecause the CHO cell has an endogenous enzyme Carboxypeptidase B (CPB)which will cleave the C-terminal lysine as long as the expressedantibody is still available intracellularly. However, this enzyme willnot cleave the lysine once the antibody is secreted into the medium.Thus, the cleavage efficiency of this endogenous CPB is based on theavailability within the cell. As such, some of the antibodies will besecreted with the lysine and some will not, and such combination willcause significant heterogeneity in the secreted product, that being someantibodies with the C-terminal lysine and some without. As therecombinant product is being used for the therapeutic use, one needs topurify to homogeneity. Thus, the recombinant products of the prior artrequires additional purification steps wherein the recombinant productneed to be treated with the enzyme CPB first and purified once againusing an additional step to remove any lysine and the enzyme CPB fromthe final product. These additional steps add a significant cost to themanufacturing process.

The present invention avoids the shortcomings of previous methods ofsynthesizing recombinant anti-CTLA-4 and anti-EGFR1 antibodies bytranscribing an expressed protein from a polynucleotide sequence lackingnucleotides for expression of the C-terminal lysine at the heavy chainof the expressed antibody.

The present invention is based on preparing chimeric/fusion proteins byexpression of polynucleotides encoding the fusion proteins thatcounteract or reverse immune tolerance of cancer cells. Cancer cells areable to escape elimination by chemotherapeutic agents or tumor-targetedantibodies via specific immunosuppressive mechanisms in the tumormicroenvironment and such ability of cancer cells is recognized asimmune tolerance. Such immunosuppressive mechanisms includeimmunosuppressive cytokines (for example, Transforming growth factorbeta (TGF-β)) and regulatory T cells and/or immunosuppressive myeloiddendritic cells (DCs). By counteracting tumor-induced immune tolerance,the present invention provides effective compositions and methods forcancer treatment, optional in combination with another existing cancertreatment. The present invention provides strategies to counteracttumor-induced immune tolerance and enhance the antitumor efficacy ofchemotherapy by activating and leveraging T cell-mediated adaptiveantitumor against resistant or disseminated cancer cells.

In another aspect, the present invention provides a molecule includingat least one targeting moiety fused with at least one immunomodulatorymoiety. The targeting moiety specifically binds a target molecule, andthe immunomodulatory moiety specifically binds one of the followingmolecules: (i) Transforming growth factor-beta (TGF-β) and or (ii)Programmed death-1 ligand 1 (PD-L1).

In a further aspect, the targeting moiety includes an antibody,including both heavy chains and light chains, wherein the antibodyspecifically binds a component of a tumor cell, tumor antigen, tumorvasculature, tumor microenvironment, or tumor-infiltrating immune cell.Notably the heavy chain and/or light chain may individually be linked toa same type immunomodulatory moiety or a separate and distinctimmunomodulatory moiety. Further, a heavy or light chain of an antibodytargeting moiety may be linked to an immunomodulatory moiety which inturn can be further linked to a second immunomodulatory moiety whereinthere is a linker between the two immunomodulatory moieties.

In a still further aspect, there is provided a chimeric polypeptide thatcomprised a tumor targeting moiety and an immunomodulatory moietycomprising a molecule that binds transforming growth factor beta(TGF-β), wherein the tumor targeting moiety is an antibody that binds toEGFR1, where in the antibody can be the full antibody, heavy chain orlight chain.

The tumor targeting moiety may include monoclonal antibodies that targeta cancer cell, including but not limited to cetuximab, trastuzumab,ritubximab, ipilimumab, tremelimumab, muromonab-CD3, abciximab,daclizumab, basiliximab, palivizumab, infliximab. gemtuzumab ozogamicin,alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab,1-131 tositumomab, efalizumab, bevacizumab, panitumumab, pertuzumab,natalizumab, etanercept, IGN101 (Aphton), volociximab (Biogen Idec andPDL BioPharm), Anti-CD80 mAb (Biogen Idec), Anti-CD23 mAb (Biogen Idel),CAT-3888 (Cambridge Antibody Technology), CDP-791 (Imclone), eraptuzumab(Immunomedics), MDX-010 (Medarex and BMS), MDX-060 (Medarex), MDX-070(Medarex), matuzumab (Merck), CP-675,206 (Pfizer), CAL (Roche), SGN-30(Seattle Genetics), zanolimumab (Serono and Genmab), adecatumumab(Sereno), oregovomab (United Therapeutics), nimotuzumab (YM Bioscience),ABT-874 (Abbott Laboratories), denosumab (Amgen), AM 108 (Amgen), AMG714 (Amgen), fontolizumab (Biogen Idec and PDL BioPharm), daclizumab(Biogent Idec and PDL BioPharm), golimumab (Centocor andSchering-Plough), CNTO 1275 (Centocor), ocrelizumab (Genetech andRoche), HuMax-CD20 (Genmab), belimumab (HGS and GSK), epratuzumab(Immunomedics), MLN1202 (Millennium Pharmaceuticals), visilizumab (PDLBioPharm), tocilizumab (Roche), ocrerlizumab (Roche), certolizumab pegol(UCB, formerly Celltech), eculizumab (Alexion Pharmaceuticals),pexelizumab (Alexion Pharmaceuticals and Procter & Gamble), abciximab(Centocor), ranibizimumab (Genetech), mepolizumab (GSK), TNX-355(Tanox), or MYO-029 (Wyeth).

In a preferred embodiment, the tumor targeting moiety is a monoclonalantibody that binds to CTLA-4 or EGFR1 generated by the methods of thepresent invention, wherein the method comprises the following steps:

a. preparing a codon optimized nucleotide sequence encoding the fusionprotein, wherein the codon optimized sequence for the antibody islacking nucleotides for expression of a lysine at the C-terminal end ofthe heavy chains of the antibody;b. cloning the optimized sequence of said fusion protein in a host cellcapable of transient or continued expression;c. growing the host cell in a media under suitable conditions forgrowing and allowing the host cell to express the fusion protein; andd. collecting secreted fusion proteins.

In yet another aspect, the immunomodulatory moiety includes a moleculethat binds TGF-β and inhibits the function thereof. Specifically theimmunomodulatory moiety includes an extracellular ligand-binding domainof Transforming growth factor-beta receptor TGF-βRII, TGF-βRIIb orTGF-βRIII In another aspect the immunomodulatory moiety includes anextracellular ligand-binding domain (ECD) of TGF-βRII

In a still further aspect, the targeting moiety includes an antibodythat specifically binds to HER2/neu, EGFR1, CD20, or cytotoxicT-lymphocyte antigen-4 (CTLA-4) and wherein the immunomodulatory moietyincludes an extracellular ligand-binding domain of TGF-βRII.

In yet another aspect, the immunomodulatory moiety includes a moleculethat specifically binds to and inhibits the activity of Programmeddeath-1 ligand 1 (PD-L 1).

In a further aspect, the targeting moiety includes an antibody, antibodyfragment, or polypeptide that specifically binds to HER2/neu, EGFR1,CD20, cytotoxic T-lymphocyte antigen-4 (CTLA-4), CD25 (1L-2a receptor;IL-2aR), or CD4 and wherein, the immunomodulatory moiety includes anextracellular ligand-binding domain or ectodomain of Programmed Death-1(PD-1).

In a still further aspect, the targeting moiety includes an antibodythat specifically binds to EGFR1 and CTLA-4, and the immunomodulatorymoiety includes a sequence from interacts with transforming growthfactor-0 (TGF-β).

In one aspect, the present invention provides for optimized genesencoding for a fusion polypeptide comprising at least one targetingmoiety and at least one immunomodulatory moiety for treating cancer in ahuman subject wherein the genes have been optimized to increaseexpression in a human subject and/or cells.

In another aspect, the present invention provides for a vectorcomprising optimized genes for treating cancer in a human subjectwherein the optimized genes have been modified to increase CG sequences.Preferably, the vector includes nucleotide sequences for encoding atleast one targeting moiety, at least one immunomodulatory moiety and alinking moiety, wherein the optimized nucleotide sequences are selectedfrom SEQ ID NOs: 1 to 7, as set forth in FIG. 2.

In yet another aspect, the present invention provides for a method oftreating cancer in a subject, the method comprising:

providing at least one recombinant vector comprising nucleotidesequences that encode at least one targeting moiety, at least oneimmunomodulatory moiety and a linking moiety positioned between thetargeting moiety and immunomodulatory moiety, wherein the nucleotidesequences are selected from SEQ ID NOs: 1 to 7; and

administering the recombinant vector to the subject under conditionssuch that said nucleotide sequences are expressed at a level whichproduces a therapeutically effective amount of the encoded fusionproteins in the subject.

In yet another aspect, the present invention provides a recombinant hostcell transfected with a polynucleotide sequence that encodes a fusionprotein peptide of the present invention, wherein the polynucleotidesequences are selected from SEQ ID NOs: 1 to 7.

In a still further aspect, the present invention contemplates a processof preparing a chimeric fusion protein of the present inventioncomprising:

transfecting a host cell with a polynucleotide sequence that encodes achimeric fusion protein to produce a transformed host cell, wherein thepolynucleotide sequence encodes at least one targeting moiety and atleast one immunomodulatory moiety, wherein the polynucleotide sequencecomprises a combination of sequences selected from SEQ ID NOs: 1 to 7;and

maintaining the transformed host cell under biological conditionssufficient for expression of the chimeric fusion protein.

In another aspect, the present invention relates to the use of achimeric fusion protein, wherein the chimeric fusion protein comprisesanti-EGFR1 linker PD1 (SEQ ID NOs: 8, 9, 10 and 11);anti-EGFR1-linker-TGFβRII (SEQ ID NOs: 8, 9, 10 and 12);Anti-CTLA-4-linker-PD1 (SEQ ID NOs: 13, 14, 10 and 11), as shown inFIGS. 3, 4 and 5 respectively, in the use of a medicament for thetreatment of cancer. Preferably, the fusion protein is expressed in ahost cell and such expressed proteins are administered in a therapeuticamount to reduce the effects of cancer in a subject in need thereof.

In a still further aspect, the present invention provides a method oftreating a neoplastic disease. The method includes administration to asubject in need thereof one or more fusion proteins of the presentinvention, in various aspects, the subject is administered one or morefusion protein of the present invention in combination with anotheranticancer therapy. In one aspect, the anticancer therapy includes achemotherapeutic molecule, antibody, small molecule kinase inhibitor,hormonal agent or cytotoxic agent. The anticancer therapy may alsoinclude ionizing radiation, ultraviolet radiation, cryoablation, thermalablation, or radiofrequency ablation.

In a preferred embodiment the therapeutically active antibody-peptidefusion proteins is a targeting antibody fused to one or moreimmunomodulating moiety that counteracts immune tolerance of a cancercell. In one aspect, the immunomodulating moiety may be linked by anamino acid spacer of sufficient length to allow bi-specific binding ofthe molecule. The immunomodulating moiety may be bound to either theN-terminus or C-terminus of the heavy chain or the N-terminus orC-terminus of the light chain of the antibody

The method of the present invention provides nucleotide sequences thatencode the therapeutically active antibody-peptide fusion proteins andsuch expression may be conducted in a transient cell line or a stablecell line. The transient expression is accomplished by transfecting ortransforming the host cell with vectors carrying the encoded fusionproteins into mammalian host cells

Once the fusion peptides are expressed, they are preferably subjected topurification and in-vitro tests to check its bi-specificity, that being,having the ability to bind to both the target moiety andimmunomodulating moiety. Such tests may include in-vitro tests such asELISA or NK/T-cell binding assays to validate bi-functional targetbinding or immune cell stimulation.

Notably once the specific fusion peptides demonstrate the desiredbi-specificity, the polynucleotide sequences encoding such fusionpeptides are selected for sub-cloning into a stable cell line for largerscale expression and purification. Such stable cell lines are previouslydisclosed, such as a mammalian cell line, including but not limited toHEK293, CHO or NSO.

In another aspect the present invention provides for a method to inhibitand/or reduce binding of PDL1 to PD1 thereby increasing immune responseagainst tumor cells, the method comprising:

a. providing a chimeric polypeptide comprising PD1 and an anti-EGFR1 oranti-CTLA-4 antibody; andb. contacting a tumor cell with the chimeric polypeptide wherein thechimeric polypeptide binds with at least PDL1 of the tumor cell.

In yet another aspect, the present invention provides for a method ofpreparing therapeutically active antibody-peptide fusion proteins, themethod comprising;

a. preparing a codon optimized sequence of the said fusion protein,wherein the codon optimized sequences for anti-EGFR1 and anti-CTLA-4antibodies are lacking nucleotides for expression of a lysine at theC-terminal end of the heavy chains of the antibodies;b. cloning the optimized sequence of said fusion protein in a host cellcapable of transient or continued expression;c. growing the host cell in a media under suitable conditions forgrowing and allowing the host cell to express the fusion protein; andd. collecting secreted fusion proteins.

In a still further aspect the present invention provides for a nucleicacid sequence encoding a chimeric fusion protein, wherein the chimericfusion protein comprises at least one targeting moiety having affinityfor a cancer cell and at least one immunomodulatory moiety thatcounteract immune tolerance of the cancer cell, wherein targeting moietyis an antibody and the nucleic acid sequence of the targeting moiety islacking nucleotides for expression of a lysine at the C-terminal end ofthe heavy chains of the antibody. The nucleic acid sequence encoding theheavy chain of the antibody preferably includes SEQ ID NO: 1 or SEQ IDNO:5. The nucleic acid sequence encoding the chimeric fusion proteinspreferably comprises a sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 4 and 7; SEQ ID NOs: 1, 2, 3 and 4; and SEQ ID NOs: 5,6, 3 and 4.

In yet another aspect the present invention provides for a method oftreating cancer in a subject, the method comprising:

a) preparing a preparing therapeutically active fusion protein, whereinthe fusion protein comprises a tumor targeting moiety and at least oneimmunomodulatory molecule, wherein the tumor targeting moiety is anantibody that binds to CTLA-4 or EGFR1 and wherein the fusion protein isprepared by the following steps:

-   -   i) preparing a codon optimized nucleotide sequence encoding the        fusion protein, wherein the codon optimized nucleotide sequence        for the antibody is lacking nucleotides for expression of a        lysine at the C-terminal end of the heavy chains of the        antibody;    -   ii) cloning the optimized sequence of said fusion protein in a        host cell capable of transient or continued expression;    -   iii) growing the host cell in a media under suitable conditions        for growing and allowing the host cell to express the fusion        protein; and    -   iv) collecting secreted fusion proteins;        b) administering a therapeutically active amount of the secreted        fusion proteins to the subject.

The fusion protein is selected from the group of amino acid sequencesconsisting of SEQ ID NOs: 15 and 9; SEQ ID NOs: 8 and 16; SEQ ID NOs: 17and 9; SEQ ID NOs: 8 and 18; SEQ ID NOs: 27 and 9; SEQ ID NOs: 8 and 28;SEQ ID NOs: 29 and 9; SEQ ID NOs: 8 and 30; SEQ ID NOs: 31 and 28; SEQID NOs: 31 and 30; SEQ ID NOs: 29 and 28; SEQ ID NOs: 29 and 30; SEQ IDNOs: 32 and 14; SEQ ID NOs: 13 and 33; SEQ ID NOs: 34 and 14; SEQ IDNOs: 13 and 35; SEQ ID NOs: 32 and 33; SEQ ID NOs: 32 and 35; SEQ IDNOs: 34 and 33 and SEQ ID NOs: 34 and 35.

In another aspect, the present invention provides for a method oftreating a neoplastic disease, the method comprising administration to asubject in need thereof one or more fusion proteins encoded by at leastone polynucleotide sequence selected from the group consisting of SEQ IDNOs: 1, 2, 4 and 7; SEQ ID NOs: 1, 2, 3 and 4; and SEQ ID NOs: 5, 6, 3and 4. Notably by using the above defined polynucleotide sequences, thefollowing combination of fusion proteins can be expressed includinganti-EGFR1 linker PD1 (SEQ ID NOs: 8, 9, 10 and 11);anti-EGFR1-linker-TGFβRII (SEQ ID NOs: 8, 9, 10 and 12); andAnti-CTLA-4-linker-PD1 (SEQ ID NOs: 13, 14, 10 and 11).

Other features and advantages of the invention will be apparent from thefollowing detailed description, drawings and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the different possibilities of lysine placement on a heavychain and such heterogeneity causing the need to provide purification.

FIG. 2 shows the optimized codon nucleotide sequences used forexpression of the antibody-peptide fusion proteins of the presentinvention, including Anti-EGFR1 heavy chain (SEQ ID NO: 1); Anti-EGFR1light chain (SEQ ID NO: 2); PD1 (SEQ ID NO: 7); Linker (SEQ ID NO: 4);Anti-CTLA-4 heavy chain (SEQ ID NO: 5); Anti-CTLA-4 light chain (SEQ IDNO: 6) and TGFβRII (SEQ ID NO: 3).

FIG. 3 shows the amino acid residues for the anti-EGFR1 linker PD1construct (SEQ ID NOs: 8, 9, 10 and 11).

FIG. 4 shows the amino acid residues for anti-EGFR1-linker-TGFβRIIconstruct (SEQ ID NOs: 8, 9, 10 and 12).

FIG. 5 shows the amino acid residues for the anti-CTLA-4-linker-PD1 (SEQID NOs: 13, 14, 10 and 11).

FIG. 6 shows the different possibilities for placement of the PD1molecule on the anti-EGFR1 antibody for FMab5, FMab6, FMab7 and FMab8.

FIG. 7 shows the amino acid sequences for Anti-EGFR1 HC-PD1+Anti-EGFR1LC wherein the PD1 molecule is connected to the C terminus of the heavychain separated by a linker and including SEQ ID NOs: 15 and 9.

FIG. 8 shows the amino acid sequences for Anti-EGFR1 HC+Anti-EGFR1LC−PD1 wherein the PD1 molecule is connected to the C terminus of thelight chain separated by a linker and including SEQ ID NOs: 8 and 16.

FIG. 9 shows the amino acid sequences for Anti-EGFR1 HC+Anti-EGFR1LC−PD1 wherein the PD1 molecule is connected to the N terminus of theheavy chain separated by a linker and including SEQ ID NOs: 17 and 9.

FIG. 10 shows the amino acid sequences for Anti-EGFR1 HC+PD1-Anti-EGFR1LC wherein the PD1 molecule is connected to the N terminus of the lightchain separated by a linker and including SEQ ID NOs: 8 and 18.

FIG. 11 shows expression constructs developed using the cDNAs as setforth in SEQ ID NOs: 1, 2 and 7.

FIG. 12 shows the different possibilities for placement of the TGFβRIImolecule on the anti-EGFR1 antibody, FMab1, FMab2, FMab3, FMab4, FMab9,FMab10, FMab11 and, FMab12.

FIG. 13 shows the amino acid sequences for Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC wherein the TGFβRII molecule is connected tothe C terminus of the heavy chain separated by a linker and includingSEQ ID NOs: 27 and 9.

FIG. 14 shows the amino acid sequences for Anti-EGFR1 HC+Anti-EGFR1LC-TGFβRII wherein the TGFβRII molecule is connected to the C terminusof the light chain separated by a linker and including SEQ ID NOs: 8 and28.

FIG. 15 shows the amino acid sequences for TGFβRII-Anti-EGFR1HC+Anti-EGFR1 LC wherein the TGFβRII molecule is connected to the Nterminus of the heavy chain separated by a linker and including SEQ IDNOs: 29 and 9.

FIG. 16 shows the amino acid sequences for Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC wherein the TGFβRII molecule is connected tothe N terminus of the light chain separated by a linker and includingSEQ ID NOs: 8 and 30.

FIG. 17 shows the amino acid sequences for Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC-TGFβRII wherein the TGFβRII molecule isconnected to the C terminus of the heavy and light chain separated by alinker and including SEQ ID NOs: 31 and 28.

FIG. 18 shows the amino acid sequences for Anti-EGFR1HC-TGFβRII+TGFβRII-Anti-EGFR1 LC wherein the TGFβRII molecule isconnected to the C terminus of the heavy chain and N terminus of thelight chain separated by a linker and including SEQ ID NOs: 31 and 30.

FIG. 19 shows the amino acid sequences for TGFβRII-Anti-EGFR1HC+Anti-EGFR1 LC-TGFβRII wherein the TGFβRII molecule is connected tothe N terminus of the heavy chain and C terminus of the light chainseparated by a linker and including SEQ ID NOs: 29 and 28.

FIG. 20 shows the amino acid sequences for TGFβRII-Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC wherein the TGFβRII molecule is connected tothe N terminus of the heavy chain and N terminus of the light chainseparated by a linker and including SEQ ID NOs: 29 and 30.

FIG. 21 shows expression constructs developed using the cDNAs as setforth in SEQ ID NOs: 1, 2 and 3.

FIG. 22 shows ProteinA purified samples analyzed on 12% reducingSDS-PAGE.

FIG. 23 shows ProteinA purified samples analyzed on 6% non-reducingSDS-PAGE.

FIG. 24 shows the different possibilities for placement of the PD1molecule on the anti-CTLA4 antibody.

FIG. 25 shows the amino acid sequences for Anti-CTLA4 HC-PD1+Anti-CTLA4LC wherein the PD1 molecule is connected to the C terminus of the heavychain separated by a linker and including SEQ ID NOs: 32 and 14.

FIG. 26 shows the amino acid sequences for Anti-CTLA4 HC+Anti-CTLA4LC-PD1 wherein the PD1 molecule is connected to the C terminus of thelight chain separated by a linker and including SEQ ID NOs: 13 and 33.

FIG. 27 shows the amino acid sequences for PD1-Anti-CTLA4 HC+Anti-CTLA4LC wherein the PD1 molecule is connected to the N terminus of the heavychain separated by a linker and including SEQ ID NOs: 34 and 14.

FIG. 28 shows the amino acid sequences for Anti-CTLA4 HC+PD1-Anti-CTLA4LC wherein the PD1 molecule is connected to the N terminus of the lightchain separated by a linker and including SEQ ID NOs: 13 and 35.

FIG. 29 shows the amino acid sequences for Anti-CTLA4 HC-PD1+Anti-CTLA4LC-PD1 wherein the PD1 molecule is connected to the C terminus of theheavy chain and light chain separated by a linker and including SEQ IDNOs: 32 and 33.

FIG. 30 shows the amino acid sequences for Anti-CTLA4HC-PD1+PD1-Anti-CTLA4 LC wherein the PD1 molecule is connected to the Cterminus of the heavy chain separated by a linker and N terminus of thelight chain including SEQ ID NOs: 32 and 35.

FIG. 31 shows the amino acid sequences for PD1-Anti-CTLA4 HC+Anti-CTLA4LC-PD1 wherein the PD1 molecule is connected to the N terminus of theheavy chain separated by a linker and C terminus of the light chainincluding SEQ ID NOs: 34 and 33.

FIG. 32 shows the amino acid sequences for PD1-Anti-CTLA4HC+PD1-Anti-CTLA4 LC wherein the PD1 molecule is connected to the Nterminus of the heavy chain separated by a linker and N terminus of thelight chain including SEQ ID NOs: 34 and 35.

FIG. 33 shows expression constructs developed using the cDNAs as setforth in SEQ ID NOs: 7, 5 and 6.

FIG. 34 shows EGFR1 target binding ELISA. The Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab binds to its immobilized targetEGFR1.

FIG. 35 shows TGFβ target binding ELISA. The Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab binds to its target TGFβ.

FIG. 36 shows Bifunctional ELISA. The anti-EGFR1 HC-TGFβRII+Anti-EGFR1LC fusion Mab binds to both its target EGFR1 and TGFβ at the same time.

FIG. 37 shows flow cytometric analysis of the binding of the anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab to EGFR1-expressing A431 cells.

FIG. 38 shows ADCC against EGFR1-expressing A-431 cells. Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab mediates ADCC againstEGFR1-expressing A-431 cells and the effect is dose dependent.

FIG. 39 shows Inhibition of proliferation assay. Anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab inhibits the proliferation ofEGFR1-expressing A-431 cells.

FIG. 40 shows EGFR1 target binding ELISA. The Anti-EGFR1 HC+Anti-EGFR1LC-TGFβRII fusion Mab binds to its immobilized target EGFR1.

FIG. 41 shows TGFβ target binding ELISA. The anti-EGFR1 HC+Anti-EGFR1LC-TGFβRII fusion Mab binds to its target TGFβ.

FIG. 42 shows Bifunctional ELISA. The anti-EGFR1 HC+Anti-EGFR1LC-TGFβRII fusion Mab binds to both its target EGFR1 and TGFβ at thesame time.

FIG. 43 shows Inhibition of proliferation assay. Anti-EGFR1HC+Anti-EGFR1 LC-TGFβRII fusion Mab inhibits the proliferation ofEGFR1-expressing A-431 cells.

FIG. 44 shows EGFR1 target binding ELISA. The TGFβRII-Anti-EGFR1HC+Anti-EGFR1 LC fusion Mab binds to its immobilized target EGFR1.

FIG. 45 shows TGFβ target binding ELISA. The TGFβRII-anti-EGFR1HC+anti-EGFR1 LC fusion Mab binds to its target TGFβ.

FIG. 46 shows Bifunctional ELISA. The TGFβRII-Anti-EGFR1 HC+Anti-EGFR1LC fusion Mab binds to both its target EGFR1 and TGFβ at the same time.

FIG. 47 shows Inhibition of proliferation assay. TGFβRII-Anti-EGFR1HC+Anti-EGFR1 LC fusion Mab inhibits the proliferation ofEGFR1-expressing A-431 cells.

FIG. 48 shows EGFR1 target binding ELISA. The Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its immobilized targetEGFR1.

FIG. 49 shows TGFβ target binding ELISA. The Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its target TGFβ.

FIG. 50 shows Bifunctional ELISA. The Anti-EGFR1 HC+TGFβRII-Anti-EGFR1LC fusion Mab binds to both its target EGFR1 and TGFβ at the same time.

FIG. 51 shows flow cytometric analysis of the binding of the Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab to EGFR1-expressing A431 cells.

FIG. 52 shows EGFR1 target binding ELISA. The Anti-EGFR1HC-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its immobilizedtarget EGFR1.

FIG. 53 shows TGFβ target binding ELISA. The Anti-EGFR1HC-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its target TGFβ.

FIG. 54 shows Bifunctional ELISA. The Anti-EGFR1HC-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab binds to both its targetEGFR1 and TGFβ at the same time.

FIG. 55 shows EGFR1 target binding ELISA. The TGFβRII-Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its immobilized targetEGFR1.

FIG. 56 shows TGFβ target binding ELISA. TGFβRII-Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab binds to its target TGFβ.

FIG. 57 shows Bifunctional ELISA. The TGFβRII-Anti-EGFR1HC+TGFβRII-Anti-EGFR1 LC fusion Mab binds to both its target EGFR1 andTGFβ at the same time.

FIG. 58 shows Bifunctional ELISA. The Anti-CTLA4 HC-PD1+Anti-CTLA4 LCfusion Mab binds to both its target CTLA4 and PDL1 at the same time.

FIG. 59 shows Bifunctional ELISA. The Anti-CTLA4 HC+Anti-CTLA4 LC-PD1fusion Mab binds to both its target CTLA4 and PDL1 at the same time.

FIG. 60 shows Bifunctional ELISA. The PD1-Anti-CTLA4 HC+Anti-CTLA4 LCfusion Mab binds to both its target CTLA4 and PDL1 at the same time.

FIG. 61 shows Bifunctional ELISA. The Anti-CTLA4 HC-PD1+PD1-Anti-CTLA4LC-PD1, Anti-CTLA4 HC-PD1+Anti-CTLA4 LC-PD1 and Anti-CTLA4HC-PD1+PD1-Anti-CTLA4 LC fusion Mabs binds to both their target CTLA4and PDL1 at the same time.

FIG. 62 shows Bifunctional ELISA. The PD1-Anti-CTLA4 HC+Anti-CTLA4LC-PD1 and PD1-Anti-CTLA4 HC+PD1-Anti-CTLA4 LC fusion Mabs binds to boththeir target CTLA4 and PDL1 at the same time.

FIG. 63 shows Bifunctional ELISA. The Anti-EGFR1 HC-PD1+Anti-EGFR1 LCfusion Mab binds to both its target EGFR and PDL1 at the same time.

FIG. 64 shows Bifunctional ELISA. The Anti-EGFR1 HC+Anti-EGFR1 LC-PD1fusion Mab binds to both its target EGFR and PDL1 at the same time.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate review of the various embodiments of theinvention and provide an understanding of the various elements andconstituents used in making and using the present invention, thefollowing terms used in the invention description have the followingmeanings.

As used herein, the terms “polypeptide,” “protein” and “peptide” areused interchangeably to denote a sequence polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). D- and L-amino acids,and mixtures of D- and L-amino acids are also included.

Chimeric polypeptide refers to an amino acid sequence having two or moreparts which generally are not found together in an amino acid sequencein nature.

The term “spacer/linker” as used herein refers to a molecule thatconnects two monomeric protein units to form a chimeric molecule andstill provides for binding of the parts to the desired receptors.Particular examples of spacer/linkers may include an amino acid spacer,wherein thee amino acid sequence can essentially be any length, forexample, as few as 5 or as many as 200 or more preferably from about 5to 30 amino acid residues.

The term “therapeutic,” as used herein, means a treatment administeredto a subject who exhibits signs of pathology for the purpose ofdiminishing or eliminating those signs.

The term “therapeutically effective amount,” as used herein means anamount of the chimeric protein that is sufficient to provide abeneficial effect to the subject to which the chimeric protein isadministered.

Another example of a modification is the addition of a heterologousdomain that imparts a distinct functionality upon the chimericpolypeptide. A heterologous domain can be any small organic or inorganicmolecule or macromolecule, so long as it imparts an additional function.Particular examples of heterologous domains that impart a distinctfunction include an amino acid sequence that imparts targeting (e.g.,receptor ligand, antibody, etc.), immunopotentiating function (e.g.,immunoglobulin, an adjuvant), enable purification, isolation ordetection (e.g., myc, T7 tag, polyhistidine, avidin, biotin, lectins,etc.).

As exemplified herein, polypeptide sequences may include substitutions,variations, or derivitizations of the amino acid sequence of one or bothof the polypeptide sequences that comprise the chimeric polypeptide, solong as the modified chimeric polypeptide has substantially the sameactivity or function as the unmodified chimeric polypeptide.

As used herein, the term “substantially the same activity or function,”when used in reference to a chimeric polypeptide so modified, means thatthe polypeptide retains most, all or more of the activity associatedwith the unmodified polypeptide, as described herein or known in theart.

Modified chimeric polypeptides that are “active” or “functional”included herein can be identified through a routine functional assay.For example, by using antibody binding assays or co-receptor bindingassays one can readily determine whether the modified chimericpolypeptide has activity. As the modified chimeric polypeptides willretain activity or function associated with unmodified chimericpolypeptide, modified chimeric polypeptides will generally have an aminoacid sequence “substantially identical” or “substantially homologous”with the amino acid sequence of the unmodified polypeptide.

As used herein, the term “substantially identical” or “substantiallyhomologous,” when used in reference to a polypeptide sequence, meansthat a sequence of the polypeptide is at least 50% identical to areference sequence. Modified polypeptides and substantially identicalpolypeptides will typically have at least 70%, alternatively 85%, morelikely 90%, and most likely 95% homology to a reference polypeptide.

As set forth herein, substantially identical or homologous polypeptidesinclude additions, truncations, internal deletions or insertions,conservative and non-conservative substitutions, or other modificationslocated at positions of the amino acid sequence which do not destroy thefunction of the chimeric polypeptide (as determined by functionalassays, e.g., as described herein). A particular example of asubstitution is where one or more amino acids are replaced by another,chemically or biologically similar residue. As used herein, the term“conservative substitution” refers to a substitution of one residue witha chemically or biologically similar residue. Examples of conservativesubstitutions include the replacement of a hydrophobic residue, such asisoleucine, valine, leucine, or methionine for another, the replacementof a polar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. Those of skill in the art will recognize the numerous aminoacids that can be modified or substituted with other chemically similarresidues without substantially altering activity.

Modified polypeptides further include “chemical derivatives,” in whichone or more of the amino acids therein have a side chain chemicallyaltered or derivatized. Such derivatized polypeptides include, forexample, amino acids in which free amino groups form aminehydrochlorides, p-toluene sulfonyl groups, carobenzoxy groups; the freecarboxy groups form salts, methyl and ethyl esters; free hydroxyl groupsthat form O-acyl or O-alkyl derivatives, as well as naturally occurringamino acid derivatives, for example, 4-hydroxyproline, for proline,5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine,and so forth. Also included are D-amino acids and amino acid derivativesthat can alter covalent bonding, for example, the disulfide linkage thatforms between two cysteine residues that produces a cyclizedpolypeptide.

As used herein, the terms “isolated” or “substantially pure,” when usedas a modifier of invention chimeric polypeptides, sequence fragmentsthereof, and polynucleotides, means that they are produced by humanintervention and are separated from their native in vivo-cellularenvironment. Generally, polypeptides and polynucleotides so separatedare substantially free of other proteins, nucleic acids, lipids,carbohydrates or other materials with which they are naturallyassociated.

Polypeptides of the present invention may be prepared by standardtechniques well known to those skilled in the art. Such techniquesinclude, but are not limited to, isolation and purification from tissuesknown to contain that polypeptide, and expression from cloned DNA thatencodes such a polypeptide using transformed cells. Chimericpolypeptides can be obtained by expression of a polynucleotide encodingthe polypeptide in a host cell, such as a bacteria, yeast or mammaliancell, and purifying the expressed chimeric polypeptide by purificationusing typical biochemical methods (e.g., immunoaffinity purification,gel purification, expression screening etc.). Other well-known methodsare described in Deutscher et al., 1990. Alternatively, the chimericpolypeptide can be chemically synthesized. Purity can be measured by anyappropriate method, e.g., polyacrylamide gel electrophoresis, andsubsequent staining of the gel (e.g., silver stain) or by HPLC analysis.

The present invention further provides polynucleotide sequences encodingchimeric polypeptides, fragments thereof, and complementary sequences.As used herein, the terms “nucleic acid,” “polynucleotide,”“oligonucleotide,” and “primer” are used interchangeably to refer todeoxyribonucleic acid (DNA) or ribonucleic (RNA), either double- orsingle-stranded, linear or circular. RNA can be unspliced or splicedmRNA, rRNA, tRNA, or antisense RNAi. DNA can be complementary DNA(cDNA), genomic DNA, or an antisense. Specifically included arenucleotide analogues and derivatives, such as those that are resistantto nuclease degradation, which can function to encode an inventionchimeric polypeptide. Nuclease resistant oligonucleotides andpolynucleotides are particularly useful for the present nucleic acidvaccines described herein.

An “isolated” or “substantially pure” polynucleotide means that thenucleic acid is not immediately contiguous with the coding sequenceswith either the 5′ end or the 3′ end with which it is immediatelycontiguous in the naturally occurring genome of the organism from whichit is derived. The term therefore includes, for example, a recombinantDNA (e.g., a cDNA or a genomic DNA fragment produced by PCR orrestriction endonuclease treatment produced during cloning), as well asa recombinant DNA incorporated into a vector, an autonomouslyreplicating plasmid or virus, or a genomic DNA of a prokaryote oreukaryote.

The polynucleotides sequences of the present invention can be obtainedusing standard techniques known in the art (e.g., molecular cloning,chemical synthesis) and the purity can be determined by polyacrylamideor agarose gel electrophoresis, sequencing analysis, and the like.Polynucleotides also can be isolated using hybridization orcomputer-based techniques that are well known in the art. Suchtechniques include, but are not limited to: (1) hybridization of genomicDNA or cDNA libraries with probes to detect homologous nucleotidesequences; (2) antibody screening of polypeptides expressed by DNAsequences (e.g., using an expression library); (3) polymerase chainreaction (PCR) of genomic DNA or cDNA using primers capable of annealingto a nucleic acid sequence of interest; (4) computer searches ofsequence databases for related sequences; and (5) differential screeningof a subtracted nucleic acid library.

The invention also includes substantially homologous polynucleotides. Asused herein, the term “homologous,” when used in reference to nucleicacid molecule, refers to similarity between two nucleotide sequences.When a nucleotide position in both of the molecules is occupied byidentical nucleotides, then they are homologous at that position.“Substantially homologous” nucleic acid sequences are at least 50%homologous, more likely at least 75% homologous, and most likely 90% ormore homologous. As with substantially homologous invention chimericpolypeptides, polynucleotides substantially homologous to inventionpolynucleotides encoding chimeric polypeptides encode polypeptides thatretain most or all of the activity or function associated with thesequence to which it is homologous. For polynucleotides, the length ofcomparison between sequences will generally be at least 30 nucleotides,alternatively at least 50 nucleotides, more likely at least 75nucleotides, and most likely 110 nucleotides or more. Algorithms foridentifying homologous sequences that account for polynucleotidesequence gaps and mismatched oligonucleotides are known in the art, suchas BLAST (see Altschul, 1990).

The polynucleotides of the present invention can, if desired: be nakedor be in a carrier suitable for passing through a cell membrane (e.g.,polynucleotide-liposome complex or a colloidal dispersion system),contained in a vector (e.g., retrovirus vector, adenoviral vectors, andthe like), linked to inert beads or other heterologous domains (e.g.,antibodies, ligands, biotin, streptavidin, lectins, and the like), orother appropriate compositions disclosed herein or known in the art.Thus, viral and non-viral means of polynucleotide delivery can beachieved and are contemplated. The polynucleotides of the presentinvention can also contain additional nucleic acid sequences linkedthereto that encode a polypeptide having a distinct functionality, suchas the various heterologous domains set forth herein.

The polynucleotides of the present invention can also be modified, forexample, to be resistant to nucleases to enhance their stability in apharmaceutical formulation. The described polynucleotides are useful forencoding chimeric polypeptides of the present invention, especially whensuch polynucleotides are incorporated into expression systems disclosedherein or known in the art. Accordingly, polynucleotides including anexpression vector are also included.

For propagation or expression in cells, polynucleotides described hereincan be inserted into a vector. The term “vector” refers to a plasmid,virus, or other vehicle known in the art that can be manipulated byinsertion or incorporation of a nucleic acid. Such vectors can be usedfor genetic manipulation (i.e., “cloning vectors”) or can be used totranscribe or translate the inserted polynucleotide (i.e., “expressionvectors”). A vector generally contains at least an origin of replicationfor propagation in a cell and a promoter. Control elements, includingpromoters present within an expression vector, are included tofacilitate proper transcription and translation (e.g., splicing signalfor introns, maintenance of the correct reading frame of the gene topermit in-frame translation of mRNA and stop codons). In vivo or invitro expression of the polynucleotides described herein can beconferred by a promoter operably linked to the nucleic acid.

“Promoter” refers to a minimal nucleic acid sequence sufficient todirect transcription of the nucleic acid to which the promoter isoperably linked (see Bitter 1987). Promoters can constitutively directtranscription, can be tissue-specific, or can render inducible orrepressible transcription; such elements are generally located in the 5′or 3′ regions of the gene so regulated.

As used herein, the term “operably linked” means that a selectedpolynucleotide (e.g., encoding a chimeric polypeptide) and regulatorysequence(s) are connected in such a way as to permit transcription whenthe appropriate molecules (e.g., transcriptional activator proteins) arebound to the regulatory sequence(s). Typically, a promoter is located atthe 5′ end of the polynucleotide and may be in close proximity of thetranscription initiation site to allow the promoter to regulateexpression of the polynucleotide.

When cloning in bacterial systems, constitutive promoters, such as T7and the like, as well as inducible promoters, such as pL ofbacteriophage gamma, plac, ptrp, ptac, may be used. When cloning inmammalian cell systems, constitutive promoters, such as SV40, RSV andthe like, or inducible promoters derived from the genome of mammaliancells (e.g., the metallothionein promoter) or from mammalian viruses(e.g., the mouse mammary tumor virus long terminal repeat, theadenovirus late promoter), may be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences of the invention.

Mammalian expression systems that utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the nucleic acid sequence may be ligatedto an adenovirus transcription/translation control complex, e.g., thelate promoter and tripartite leader sequence. Alternatively, thevaccinia virus 7.5K promoter may be used (see Mackett 1982; Mackett1984; Panicali 1982).

For yeast expression, a number of vectors containing constitutive orinducible promoters may be used (see Ausubel 1988; Grant 1987; Glover1986; Bitter 1987; and Strathem 1982). The polynucleotides may beinserted into an expression vector for expression in vitro (e.g., usingin vitro transcription/translation kits, which are availablecommercially), or may be inserted into an expression vector thatcontains a promoter sequence that facilitates expression in eitherprokaryotes or eukaryotes by transfer of an appropriate nucleic acidinto a suitable cell, organ, tissue, or organism in vivo.

As used herein, a “transgene” is any piece of a polynucleotide insertedby artifice into a host cell, and becomes part of the organism thatdevelops from that cell. A transgene can include one or more promotersand any other DNA, such as introns, necessary for expression of theselected DNA, all operably linked to the selected DNA, and may includean enhancer sequence. A transgene may include a polynucleotide that ispartly or entirely heterologous (i.e., foreign) to the transgenicorganism, or may represent a gene homologous to an endogenous gene ofthe organism. Transgenes may integrate into the host cell's genome or bemaintained as a self-replicating plasmid.

As used herein, a “host cell” is a cell into which a polynucleotide isintroduced that can be propagated, transcribed, or encoded polypeptideexpressed. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell, since there may be mutations that occur during replication. Hostcells include but are not limited to bacteria, yeast, insect, andmammalian cells. For example, bacteria transformed with recombinantbacteriophage polynucleotide, plasmid nucleic acid, or cosmid nucleicacid expression vectors; yeast transformed with recombinant yeastexpression vectors; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV), or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid), insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus), or animal cell systems infectedwith recombinant virus expression vectors (e.g., retroviruses,adenovirus, vaccinia virus), or transformed animal cell systemsengineered for stable expression.

As used herein, the term “transformation” means a genetic change in acell following incorporation of a polynucleotide (e.g., a transgene)exogenous to the cell. Thus, a “transformed cell” is a cell into which,or a progeny of which, a polynucleotide has been introduced by means ofrecombinant techniques. Transformation of a host cell may be carried outby conventional techniques known to those skilled in the art. When thehost cell is a eukaryote, methods of DNA transformation include, forexample, calcium phosphate, microinjection, electroporation, liposomes,and viral vectors. Eukaryotic cells also can be co-transformed withinvention polynucleotide sequences or fragments thereof, and a secondDNA molecule encoding a selectable marker, as described herein orotherwise known in the art. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells, and express theprotein (see Gluzman 1982). When the host is prokaryotic (e.g., E.coli), competent cells that are capable of DNA uptake can be preparedfrom cells harvested after exponential growth phase and subsequentlytreated by the CaCl₂) method using procedures well-known in the art.Transformation of prokaryotes also can be performed by protoplast fusionof the host cell.

Chimeric polypeptides, polynucleotides, and expression vectorscontaining same of the present invention can be encapsulated withinliposomes using standard techniques and introduced into cells or wholeorganisms. Cationic liposomes are preferred for delivery ofpolynucleotides. The use of liposomes for introducing variouscompositions in vitro or in vivo, including proteins andpolynucleotides, is known to those of skill in the art.

Liposomes can be targeted to a cell type or tissue of interest by theaddition to the liposome preparation of a ligand, such as a polypeptide,for which a corresponding cellular receptor has been identified.Monoclonal antibodies can also be used for targeting; many suchantibodies specific for a wide variety of cell surface proteins areknown to those skilled in the art and are available. The selected ligandis covalently conjugated to a lipid anchor in either preformed liposomesor are incorporated during liposome preparation (see Lee 1994 and Lee1995).

As the chimeric polypeptides or polynucleotides of the present inventionwill be administered to humans, the present invention also providespharmaceutical formulations comprising the disclosed chimericpolypeptides or polynucleotides. The compositions administered to asubject will therefore be in a “pharmaceutically acceptable” or“physiologically acceptable” formulation.

As used herein, the terms “pharmaceutically acceptable” and“physiologically acceptable” refer to carriers, diluents, excipients,and the like that can be administered to a subject, preferably withoutexcessive adverse side effects (e.g., nausea, headaches, etc.). Suchpreparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions, orsuspensions, including saline and buffered media. Vehicles includesodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobial,anti-oxidants, chelating agents, and inert gases and the like. Variouspharmaceutical formulations appropriate for administration to a subjectknown in the art are applicable in the methods of the invention (e.g.,Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.,Easton, Pa. (1990); and The Merck Index, 12th ed., Merck PublishingGroup, Whitehouse, N.J. (1996)).

Controlling the duration of action or controlled delivery of anadministered composition can be achieved by incorporating thecomposition into particles or a polymeric substance, such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate,methylcellulose, carboxymethylcellulose, protamine sulfate orlactide/glycolide copolymers, polylactide/glycolide copolymers, orethylenevinylacetate copolymers. The rate of release of the compositionmay be controlled by altering the concentration or composition of suchmacromolecules. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, beads, and lipid-based systems,including oil-in-water emulsions, micelles, mixed micelles, andliposomes.

The compositions administered by a method of the present invention canbe administered parenterally by injection, by gradual perfusion overtime, or by bolus administration or by a microfabricated implantabledevice. The composition can be administered via inhalation,intravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity (e.g., vaginal or anal), transdermally, topically, orintravascularly. The compositions can be administered in multiple doses.An effective amount can readily be determined by those skilled in theart.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Other features and advantages of theinvention will be apparent from the following detailed description, andfrom the claims. The invention is further described in the followingexamples, which do not limit the scope of the invention(s) described inthe claims.

Examples 1. Anti-EGFR1-PD1 Fusion Protein Constructs for Cancer Targets

Anti-EGFR (Cetuximab) has been approved for squamous Head and NeckCancer (locally or regionally advanced in combination with radiotherapyand metastatic after platinum based therapy) and EGFR expressingmetastatic colorectal cancer (monotherapy in patients after failure ofboth oxaliplatin and irinotecan based chemo or in patients intolerant toirinotecan based chemo). Not applicable for colonrectal cancer (CRC)patients having K-RAS mutations.

Across various studies about 55-60% of mCRC patients respond tocetuximab in first line setting, however, this response too is transient(progression free survival (PFS) advantage of 1.5-2 mths) (EPAR).Significant numbers of patients either do not respond to cetuximab orbecome resistant to therapy. In the recurrent metastatic head and neckcancer, only 35% patients respond to cetuximab with chemo with only 2-3month overall survival (OS) and (PFS) advantage.

Clearly, a significant unmet need exists to improve efficacy ofcetuximab therapy in both these indications. Moreover, EGFR is alsoexpressed in gastric cancer, non-small cell lung cancer (NSCLC) andpancreatic cancers. However, cetuximab has failed to prove anysignificant benefit in these indications over standard of care. Thus,the present invention provides for improvement by combining cetuximabwith an immunomodulatory therapy.

Programmed death-1 (PD-1) is an inhibitory receptor expressed on T cellsafter activation. It has been shown to down-regulate T-cell activityupon binding its ligand PD-L1 on APCs. Many tumors constitutivelyexpress PD-L1 and its' over expression has been associated with impairedtumor immunity, more aggressive disease and decreased survival (seeThompson 2004). Till date PD-L1 expression has been demonstrated tocorrelate with poor prognosis in patients with renal cell carcinoma(RCC), ovarian cancer and melanoma. Immunohistochemical analysis offreshly isolated tumor samples from patients with ovarian, lung, andbreast cancers, renal cell carcinoma, squamous cell carcinoma of thehead and neck, esophageal carcinoma, glioblastoma, thymoma, coloncarcinoma, pancreatic and melanoma found that the vast majority expressB7-H1 (see Flies 2011; Nomi 2007). Several pre-clinical studies havedemonstrated increased tumor rejection by blocking PD1-PDL1 interaction.Recently, anti-PD1 and PD-L1 based therapies have demonstratedconsiderable activity in melanoma and some other solid tumors confirmingtheir application as one of the most promising anti-cancer therapies.

Cetuximab based therapy may be improved upon by combining it withimmunomodulation to remove immunosuppressive environment or delay thedevelopment of resistance. Moreover, patients who develop resistance tocetuximab due to mutations in the downstream pathways may still benefitfrom Anti-EGFR1-PD-1 since the fusion protein of the present inventionbinds to the EGFR receptor and negates the PD-L1 expressed by thetumors, allowing T cells to mount an anti-tumor response. Accordingly,the fusion proteins of the present invention can bind to both EGFR andPD-L1 on the surface of the tumor cells.

The anti-EGFR1-PD1 fusion protein constructs of the present inventionmay be used in colorectal cancer, squamous head and neck cancer,non-small cell lung cancer, gastric cancer and pancreatic cancer.

Design and selection of the molecules:

The antibody fusion molecules of the present invention have dueltherapeutic properties. On one hand the molecule retains the completeactivity of the Anti-EGFR1 (Cetuximab) and in parallel, it has the PD-L1receptor binding activity in the tumor environment. The new molecules ofthe Anti-EGFR1-PD1 fusion proteins that were developed for the cancertherapies herein are devoid of the amino acid lysine ‘K’ from theC-terminus of heavy chain for the reasons described above. The mainobjective of the fusion protein design is to keep the anti-EGFR1molecule intact along with its function unaffected and allows fusion ofthe PD1 molecule to the various location on the anti-EGFR1 antibody.That being, fusion to the HC C-terminus, LC C-terminus, HC N-terminus,and or LC N-terminus and double fusions on both the chains as shown inFIG. 6.

The following constructs were designed.

TABLE 1 Constructs. no. Fusion mAbs name FMab5 Anti-EGFR1 HC-PD1 +Anti-EGFR1 LC FIG. 6 (AA sequences in FIG. 7, SEQ ID NO: 15 and 9) FMab6Anti-EGFR1 HC + Anti-EGFR1 LC -PD1 FIG. 6 (AA sequences in FIG. 8, SEQID NO: 8 and 16) FMab7 PD1-Anti-EGFR1 HC + Anti-EGFR1 LC FIG. 6 (AAsequences in FIG. 9, SEQ ID NO: 17 and 9) FMab8 Anti-EGFR1 HC +PD1-Anti-EGFR1 LC FIG. 6 (AA sequences in FIG. 10, SEQ ID NO: 8 and 18)

Expression of the above fusion constructs in CHO cells:

The codon-optimized nucleotide sequences of the Anti-EGFR1-PD1individual domains were optimized for expression in CHO cells. Suchoptimized sequences (SEQ ID NOs: 1, 2, 7, and 4) were assemble in amammalian expression vector with help of primers described in Table 2:

TABLE 2 FMAB7FP1 AGA TAT CGC CAC CAT GAT GTC CTT CGT G SEQ ID NO: 19FMAB7FP2 GGC GGC GGA GGC TCT CAG GTG CAG CTG AAG CAG TC SEQ ID NO: 20FMAB7RP1 AGT ATA CTC AGC CGG GGG ACA GAG A SEQ ID NO: 21 FMAB7RP2TTC AGC TGC ACC TGA GAG CCT CCG CCG CCA CTT C SEQ ID NO: 22 FMAB7LCRPATT AAT TAA TCA ACA CTC GCC CCG GTT GAA GGA CT SEQ ID NO: 23 FMAB6FP2CTC TGT CCC CCG GCG GCG GCG GAG GAT CTG GCG GA SEQ ID NO: 24 FMAB6RP2GAT CCT CCG CCG CCG CCG GGG GAC AGA GAC AGG GA SEQ ID NO: 25 FMAB6RP1AGT ATA CTC ACA CCA GGG TCT GGA AC SEQ ID NO: 26

Using the shown above cDNA primers set, constructs were assembled asshown in FIG. 11.

2. Anti-EGFR1-TGFβRII Fusion Proteins for Treatment of Cancer.

High levels of TGFβ are produced by many types of tumors, includingmelanomas and cancers of the breast, colon, esophagus, stomach, liver,lung, pancreas, and prostate, as well as hematologic malignancies (seeTeicher 2001; Dong 2006). TGFβ is known to be immunosuppressive for Tcells and NK cells through blocking of IL-2 and other mechanisms,including generation of T-regs. Several lines of evidence suggest thatnegating TGFβ activity may enhance anti-tumor effects of T cells(Wrzesinski 2007). Moreover, TGFβ can foster tumor growth throughepithelial to mesenchymal transition and promoting angiogenesis. TGFβexpression is also associated with poor prognosis in patients andearlier recurrence. However, considering the pleotropic effects of TGFβin controlling the immune response, it has been shown that generalizedblocking of TGFβ activity may result in widespread auto-inflammatoryactivity. Hence, localized depletion of TGFβ in the tumor vicinity maybe an alternative way to modulate immunosuppressive environment.Anti-EGFR1-TGFβRII fusion protein of the present invention binds to EGFRon the tumor cells and ties up the TGFβ around the tumor to enhanceimmune response against tumor cells.

Design and selection of the molecules:

The objective is to design the antibody fusion molecules which have dueltherapeutic properties. On one hand the molecule should retain thecomplete activity of the Anti-EGFR1 (cetuximab) and in parallel; itshould have the TGFβ binding activity in the tumor environment. Theamino acid sequence of the Anti-EGFR1 IgG molecule was retainedexcepting that the lysine was not expressed at the C-terminus of theheavy chain. Both single and double fusion and expression levels areshown in Table 3, wherein TGFβRII was fused with Anti-EGFR1.

TABLE 3 Construct. no. Fusion Mabs name FMab1 Anti-EGFR1 HC-TGFβRII +Anti-EGFR1 LC FIG. 12 (AA sequences in FIG. 13, SEQ ID NO: 27 and 9)FMab2 Anti-EGFR1 HC + Anti-EGFR1 LC -TGFβRII FIG. 12 (AA sequences inFIG. 14, SEQ ID NO: 8 and 28) FMab3 TGFβRII-Anti-EGFR1 HC + Anti-EGFR1LC FIG. 12 (AA sequences in FIG. 15, SEQ ID NO: 29 and 9) FMab4Anti-EGFR1 HC + TGFβRII-Anti-EGFR1 LC FIG. 12 (AA sequences in FIG. 16,SEQ ID NO: 8 and 30) FMab9 Anti-EGFR1 HC-TGFβRII + Anti-EGFR1 LC-TGFβRII) FIG. 12 (AA sequences in FIG. 17, SEQ ID NO: 31 and 28) FMab10Anti-EGFR1 HC-TGFβRII + TGFβRII-Anti-EGFR1 LC FIG. 12 (AA sequences inFIG. 18, SEQ ID NO: 31 and 30) FMab11 TGFβRII-Anti-EGFR1 HC + Anti-EGFR1LC -TGFβRII FIG. 12 (AA sequences in FIG. 19, SEQ ID NO: 29 and 28)FMab12 TGFβRII-Anti-EGFR1 HC + TGFβRII-Anti-EGFR1 LC FIG. 12 (AAsequences in FIG. 20, SEQ ID NO: 29 and 30)

Expression of the above fusion constructs in CHO cells:

The codon-optimized nucleotide sequences of the Anti-EGFR1-TGFβRIIindividual domains were optimized for expression in CHO cells. Suchsequences (SEQ ID NOs: 1, 2, 4, and 3) were assemble in a mammalianexpression vector. The expression constructs are set forth in FIG. 21.

Transfection of the above vectors combination to obtain the desired cellline:

The expression constructs developed above were transfected in thefollowing combination, as set forth in Table 4, into CHO cells toproduce the following fusion proteins using the constructs as defined inFIG. 21.

TABLE 4 Expression constructs Cell line Titer Sl. No. Fusion proteinName combination transfected used g/L FMab1 Anti-EGFR1 HC-TGFβRII +Expression constructs # CHO 0.11 Anti-EGFR1 LC (HC-C-terminus) 2C and 3C FMab2 Anti-EGFR1 HC + Anti-EGFR1 Expression constructs # CHO 0.10 LC -TGFβRII 1 C and 4 C FMab3 TGFβRII -Anti-EGFR1 HC + Expression constructs# CHO 0.09 Anti-EGFR1 LC 2 C and 5 C FMab4 Anti-EGFR1 HC + TGFβRII -Expression constructs # CHO 0.08 Anti-EGFR1 LC 1 C and 6 C FMab9Anti-EGFR1 HC-TGFβRII + Expression constructs # CHO ND/VL Anti-EGFR1 LC-TGFβRII 3 C and 4 C FMab10 Anti-EGFR1 HC-TGFβRII + Expressionconstructs # CHO 0.06 TGFβRII-Anti-EGFR1 LC 3 C and 6 C FMab11TGFβRII-Anti-EGFR1 HC + Expression constructs # CHO ND/VL Anti-EGFR1LC - TGFβRII 4 C and 5 C FMab12 TGFβRII-Anti-EGFR1 HC + Expressionconstructs # CHO 0.06 TGFβRII-Anti-EGFR1 LC 5 C and 6 C

Purification of the Fusion Mabs supernatants using Protein A column:

The fusion monoclonal antibodies (Mabs) using recombinant proteinproducing CHO cell culture supernatant.

Procedure:

The procedure describes in detail the small scale purification processof IgG using C10/10 or XK26 column and using Mab Select Xtra affinityresin. The samples generated by this protocol can be used for variousanalysis

Process flow:

-   -   The culture supernatant secreted from recombinant cell line        producing monoclonal antibodies or fusion monoclonal antibodies        under sterile conditions were tested for titer and endotoxins;    -   The affinity chromatography using Mab Select Xtra Protein A        resin was washed and equilibrated with binding buffer;    -   The pH of the supernatant was adjusted using 0.5M phosphate to        the same pH has the column;    -   The supernatant was allowed to bind to the column/pass through        the column at the flow rate of 0.5 ml/minute to achieve the        maximum binding;    -   All the fusion Mabs binds through the Fc region and rest of the        impurities passed pass through as flow through;    -   The column was washed with equilibration buffer;    -   The bound fusion Mabs were eluted using 0.1 M glycine pH 3.0;    -   The eluted proteins were adjusted back to neutral pH or the        stable formulation pH;    -   The purified proteins were stored at −20° C. or at 2-8° C.        depending on the stability.

Analysis of Protein A purified Fusion Mabs using SDS PAGE:

The transfected supernatants obtained were purified using proteinAaffinity column. Later these were analyzed on reducing and non-reducingSDS-PAGE to find out the integrity of the molecule, as shown in FIG. 22,where in the proteinA purified samples were analyzed on 12% reducingSDS-PAGE. As expected all the fusion partners are giving the expectedpattern on SDS-PAGE. The LC fusion and HC are running closely but thebands are separated. This higher mobility may be due to the 8N-glycosylation sites (TGBRII 3*2=6+2 on LC)

FIG. 23 shows the results of the ProteinA purified samples that wereanalyzed on 6% non-reducing SDS-PAGE and although the amino acidcomposition is same, there is a difference in mobility. It may be due tothe variable levels of glycosylation pattern based on the TGFβRIIposition and access in the molecule.

3. Anti-CTLA4-PD1 Fusion Protein Constructs for Cancer Targets.

Immunohistochemical analysis of freshly isolated tumor samples frompatients with ovarian, lung, and breast cancers, renal cell carcinoma,squamous cell carcinoma of the head and neck, esophageal carcinoma,glioblastoma, thymoma, colon carcinoma, pancreatic and melanoma foundthat the vast majority express B7-H1 (see Flies 2011; Nomi 2007).Several pre-clinical studies have demonstrated increased tumor rejectionby blocking PD1-PDL1 interaction. Recently, anti-PD1 and PD-L1 basedtherapies have demonstrated considerable activity in melanoma and someother solid tumors confirming their application as one of the mostpromising anti-cancer therapies.

Although, anti-CTLA4 may allow co-stimulation of T cells, they may stillbe inhibited by PD-L1-PD-1 interaction. This may be one of the reasonsfor only a minority of patients having response to anti-CTLA4 antibody.Fusion antibody of both anti-CTLA4 and PD1 are more efficacious thaneither agent alone since anti-CTLA4 allows T cell co-stimulation whereasPD1 binds to PD-L1 on tumor cells to negate the immunosuppression of Tcells in tumor microenvironment. This may even be safer than theanti-CTLA4 because the lone use of anti-CTLA4 has led to immunebreakthrough adverse events.

Design and selection of the molecules:

The objective is to design the antibody fusion molecules which have dueltherapeutic properties. On one hand the molecule should retain thecomplete activity of the anti-CTLA4 (Ipilimumab) and in parallel; itshould have the PD L 1 receptor binding activity in the tumorenvironment. The complete amino acid sequence of the anti-CTLA4 IgGmolecule was used except the removal of the lysine at the C-terminus ofthe heavy chain. A 15 amino acid linker was positioned between the PD1and Anti-CTLA4. The following combinations of constructs, as set forthin Table 5, were designed as shown in FIG. 24. The details of the abovefusion protein constructs are given below.

TABLE 5 Constructs. no. Fusion Mabs name FMab13 Anti-CTLA4 HC-PD1 +Anti-CTLA4 LC FIG. 21 (AA sequences in FIG. 25, SEQ ID NO: 32 and 14)FMab14 Anti-CTLA4 HC + Anti-CTLA4 LC-PD1 FIG. 21 (AA sequences in FIG.26, SEQ ID NO: 13 and 33) FMab15 PD1-Anti-CTLA4 HC + Anti-CTLA4 LC FIG.21 (AA sequences in FIG. 27, SEQ ID NO: 34 and 14) FMab16 Anti-CTLA4HC + PD1-Anti-CTLA4 LC FIG. 21 (AA sequences in FIG. 28, SEQ ID NO: 13and 35) FMab17 Anti-CTLA4 HC-PD1 + Anti-CTLA4 LC-PD1 FIG. 21 (AAsequences in FIG. 29, SEQ ID NO: 32 and 33) FMab18 Anti-CTLA4 HC-PD1 +PD1-Anti-CTLA4 LC FIG. 21 (AA sequences in FIG. 30, SEQ ID NO: 32 and35) FMab19 PD1-Anti-CTLA4 HC + Anti-CTLA4 LC-PD1 FIG. 21 (AA sequencesin FIG. 31, SEQ ID NO: 34 and 33) FMab20 PD1-Anti-CTLA4 HC +PD1-Anti-CTLA4 LC FIG. 21 (AA sequences in FIG. 32, SEQ ID NO: 34 and35)

Expression of the above fusion constructs in CHO cells:

The complete nucleotide sequence of the Anti-CTLA4-PD1 individualdomains were codon optimized for expression in CHO cells (SEQ ID NOs: 7,4, 5 and 6). The cDNAs were synthesized. The constructs were assembledin mammalian expression vectors. The expression of anti-CTLA4-PD1 fusionproteins using the constructs as set forth in FIG. 33.

The expression constructs developed and shown in FIG. 33 weretransfected in the following combination into CHO cells (Table 6) toproduce the following fusion proteins. The titer obtained for eachconstructs are mentioned in the last column.

TABLE 6 Expression constructs Cell line Titer Sl. No. Fusion proteinName combination transfected used g/L FMab Anti-CTLA4 HC-PD1 +Expression constructs # CHO 0.175 13 Anti-CTLA4 LC 2C and 3 C FMabAnti-CTLA4 HC + Expression constructs # CHO 0.221 14 Anti-CTLA4 LC-PD1 1C and 4C FMab PD1-Anti-CTLA4 HC + Expression constructs # CHO 0.029 15Anti-CTLA4 LC 2 C and 5 C FMab Anti-CTLA4 HC + PD1- Expressionconstructs # CHO 0.021 16 Anti-CTLA4 LC 1 C and 6C FMab Anti-CTLA4HC-PD1 + Expression constructs # CHO 0.137 17 Anti-CTLA4 LC-PD1 3 C and4C FMab Anti-CTLA4 HC-PD1 + Expression constructs # CHO 0.012 18PD1-Anti-CTLA4 LC 3 C and 6C FMab PD1-Anti-CTLA4 HC + Expressionconstructs # CHO 0.029 19 Anti-CTLA4 LC-PD1 4 C and 5 C FMabPD1-Anti-CTLA4 HC + Expression constructs # CHO 0.014 20 PD1-Anti-CTLA4LC 5 C and 6C

Purification of and characterization of Fusion proteins:

The procedure describes the use of small scale purification process ofIgG using C10/10 or XK26 column and using Mab Select Xtra affinityresin. The samples generated by this protocol can be used for variousanalysis.

Process flow:

-   -   The culture supernatant secreted from recombinant cell line        producing monoclonal antibodies or fusion monoclonal antibodies        under sterile conditions were tested for titer and endotoxins;    -   The affinity chromatography using Mab Select Xtra ProteinA resin        was washed and equilibrated with binding buffer;    -   The pH of the supernatant was adjusted using 0.5M phosphate to        the same PH has the column;    -   The supernatant was allowed to bind to the column/pass through        the column at the flow rate of 0.5 ml/minute to achieve the        maximum binding;    -   All the fusion Mabs binds through the Fc region and rest of the        impurities passed through as flow through;    -   The column was washed with equilibration buffer;    -   The bound fusion Mabs were eluted using 0.1 M glycine pH 3.0;    -   The eluted proteins were adjusted back to neutral pH or the        stable formulation pH; and    -   The purified proteins are stored at −20° C. or at 2-8° C.        depending on the stability.

4. Anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC (Fmab 1)

Binding ELISAs-Procedure:

The fusion Mab was tested for its ability to bind to its targets inthree different ELISAs: 1) EGFR1 target-binding ELISA, 2) TGFβ-targetbinding ELISA and 3) Bifunctional ELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate was incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr. The platewas washed and streptavidin-HRP was added and the plate incubated atroom temperature for 1 hr. After washing, TMB substrate solution wasadded and the reaction stopped with 1N H₂SO₄. The absorbance wasmeasured at 450 nm on a BioTek Synergy H4 Hybrid reader.

Results:

The binding of the anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion Mab toboth the targets EGFR1 (FIG. 34) and TGFβ (FIG. 35) was comparable withanti-EGFR1-TGFβRII. The anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion Mabwas also tested in a bifunctional ELISA to determine whether theanti-EGFR1 and TGFβRII domains of the Mab can bind to their respectivetargets without interfering with each other. As seen in FIG. 36, theanti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion Mab binds to both itstargets, suggesting that there is no interference in binding to eithertarget dues to the construction of the fusion Mab.

Binding to Cells Expressing EGFR1:

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized and harvested. The cells were stained withdifferent dilutions of the anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusionMab or control Ig at 2-8° C. for 30 minutes. The cells were washed andincubated with anti-Human IgG-FITC conjugate at 2-8° C. for 30 minutes.After washing, the cells were analyzed on a flow cytometer. Live cellswere gated based on their FSC vs SSC profiles. The total MFI for thegated population were recorded.

Results:

The anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion binds to EGFR-expressingA-431 cells in a dose dependent manner (FIG. 37). The binding ofanti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC is comparable to the binding ofanti-EGFR1-TGFβRII.

Antibody-dependent cytotoxicity Activity

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized, harvested and plated into 96-well plates.The cells were labeled with different dilutions of the anti-EGFR1HC-TGFβRII+Anti-EGFR1 LC fusion Mab or control Ig at 2-8° C. for 30minutes. The labeled cells were co-incubated with freshly isolated humanPBMC at 37° C., 5% CO₂ for 24 hours. Cytotoxicity was measured using theCyto-Tox-Glo cytotoxicity assay kits.

Results:

The anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion mediates ADCC ofEGFR-expressing A-431 cells by human PBMC effector cells. The ADCC isdose dependent (FIG. 38). These results suggest that the Fc portion ofthe fusion Mab is intact and functional.

Inhibition of Proliferation

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized, harvested and plated into 96-well plates.Different dilutions of the anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusionMab or control Ig were added to the cells. The plates were incubated at37° C., 5% CO₂ for three days. On the third day, cell proliferation wasmeasured by the AlamarBlue method.

Results:

Anti-EGFR antibodies such as Cetuximab are known to inhibit theproliferation of EGFR1-expressing cells. As seen in FIG. 39, theanti-EGFR portion of the anti-EGFR1 HC-TGFβRII+Anti-EGFR1 LC fusion Mabis intact and has anti-proliferative activity.

5. Anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII (Fmab2)

Binding ELISAs: Procedure:

The anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusion Mab was tested for itsability to bind to its targets in three different ELISAs: 1) EGFR1target-binding ELISA, 2) TGFβ-target binding ELISA and 3) BifunctionalELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate is incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr. The platewas washed and streptavidin-HRP was added and the plate incubated atroom temperature for 1 hr. After washing, TMB substrate solution wasadded and the reaction stopped with 1N H₂SO₄. The absorbance wasmeasured at 450 nm on a BioTek Synergy H4 Hybrid reader.

Results:

The binding of anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusion Mab to boththe targets EGFR1 (FIG. 40) and TGFβ (FIG. 41) was comparable toanti-EGFR1-TGFβRII. The anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusion Mabwas also tested in a bifunctional ELISA to determine whether theanti-EGFR1 and TGFβRII domains of the Mab can bind to their respectivetargets without interfering with each other. As seen in FIG. 42, theanti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusion Mab binds to both itstargets, suggesting that there is no interference in binding to eithertarget dues to the construction of the fusion Mab.

Inhibition of Proliferation

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized, harvested and plated into 96-well plates.Different dilutions of the anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusionMab or control Ig were added to the cells. The plates were incubated at37° C., 5% CO₂ for three days. On the third day, cell proliferation wasmeasured by the AlamarBlue method.

Results:

Anti-EGFR antibodies such as Cetuximab are known to inhibit theproliferation of EGFR1-expressing cells. As seen in FIG. 43, theanti-EGFR portion of the anti-EGFR1 HC+Anti-EGFR1 LC-TGFβRII fusion Mabis intact and has anti-proliferative activity.

6. TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC (Fmab 3)

Binding ELISAs: Procedure:

The fusion Mab was tested for its ability to bind to its targets inthree different ELISAs: 1) EGFR1 target-binding ELISA, 2) TGFβ-targetbinding ELISA and 3) Bifunctional ELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate was incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr. The platewas washed and streptavidin-HRP was added and the plate incubated atroom temperature for 1 hr. After washing, TMB substrate solution wasadded and the reaction stopped with 1N H2504. The absorbance wasmeasured at 450 nm on a BioTek Synergy H4 hybrid reader.

Results:

The binding of the TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusion Mab toboth the targets EGFR1 (FIG. 44) and TGFβ (FIG. 45) was comparable toanti-EGFR1-TGFβRII. The TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusion Mabwas also tested in a bifunctional ELISA to determine whether theanti-EGFR1 and TGFβRII domains of the Mab can bind to their respectivetargets without interfering with each other. As seen in FIG. 46, thebinding of TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusion Mab is reduced ascompared to anti-EGFR1-TGFβRII, suggesting that there is someinterference in binding to either target due to the construction of thefusion Mab.

Binding to Cells Expressing EGFR1:

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized and harvested. The cells were stained withdifferent dilutions of the TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusionMab or control Ig at 2-8° C. for 30 minutes. The cells were washed andincubated with anti-Human IgG-FITC conjugate at 2-8° C. for 30 minutes.After washing, the cells were analyzed on a flow cytometer. Live cellswere gated based on their FSC vs SSC profiles. The total MFI for thegated population were recorded.

Results:

The TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusion binds to EGFR-expressingA-431 cells in a dose dependent manner. The binding ofTGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC is comparable to the binding ofanti-EGFR1-TGFβRII.

Inhibition of Proliferation

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized, harvested and plated into 96-well plates.Different dilutions of the TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusionMab or control Ig were added to the cells. The plates were incubated at37° C., 5% CO₂ for three days. On the third day, cell proliferation wasmeasured by the AlamarBlue method.

Results:

Anti-EGFR antibodies such as Cetuximab are known to inhibit theproliferation of EGFR1-expressing cells. As seen in FIG. 47, theanti-EGFR portion of the TGFβRII-Anti-EGFR1 HC+Anti-EGFR1 LC fusion Mabis intact and has anti-proliferative activity.

7. Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC (Fmab 4)

Binding ELISAs: Procedure:

The fusion Mab was tested for its ability to bind to its targets inthree different ELISAs: 1) EGFR1 target-binding ELISA, 2) TGFβ-targetbinding ELISA and 3) Bifunctional ELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate was incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr. The platewas washed and streptavidin-HRP was added and the plate incubated atroom temperature for 1 hr. After washing, TMB substrate solution wasadded and the reaction stopped with 1N H₂SO₄. The absorbance wasmeasured at 450 nm on a BioTek Synergy H4 hybrid reader.

Results:

The binding of the Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab toboth the targets EGFR1 (FIG. 48) and TGFβ (FIG. 49) was comparable withanti-EGFR1-TGFβRII. The Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mabwas also tested in a bifunctional ELISA to determine whether theanti-EGFR1 and TGFβRII domains of the Mab can bind to their respectivetargets without interfering with each other. As seen in FIG. 50, thebinding of Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab is reduced ascompared to anti-EGFR1-TGFβRII, suggesting that there is someinterference in binding to either target due to the construction of thefusion Mab.

Binding to Cells Expressing EGFR1:

Procedure:

A-431 cells were grown in flasks until they reached 70-80% confluency.The cells were trypsinized and harvested. The cells were stained withdifferent dilutions of Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab orcontrol Ig at 2-8° C. for 30 minutes. The cells were washed andincubated with anti-Human IgG-FITC conjugate at 2-8° C. for 30 minutes.After washing, the cells were analyzed on a flow cytometer. Live cellswere gated based on their FSC vs SSC profiles. The total MFI for thegated population were recorded.

Results:

The Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion binds to EGFR-expressingA-431 cells in a dose dependent manner (FIG. 51). The binding ofAnti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC is reduced compared to the bindingof anti-EGFR1-TGFβRII.

8. Anti-EGFR1 HC-TGFβRII+TGFβRII-Anti-EGFR1 LC (Fmab 10)

Binding ELISAs: Procedure:

The fusion Mab was tested for its ability to bind to its targets inthree different ELISAs: 1) EGFR1 target-binding ELISA, 2) TGFβ-targetbinding ELISA and 3) Bifunctional ELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate was incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr.

The plate was washed and streptavidin-HRP was added and the plateincubated at room temperature for 1 hr. After washing, TMB substratesolution was added and the reaction stopped with 1N H₂SO₄. Theabsorbance was measured at 450 nm on a BioTek Synergy H4 hybrid reader.

Results:

The binding of Anti-EGFR1 HC-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab tothe target EGFR1 was slightly reduced (FIG. 52) but was higher for TGFβ(FIG. 53) when compared to the binding of anti-EGFR1-TGFβRII. TheAnti-EGFR1-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab was also tested in abifunctional ELISA to determine whether the anti-EGFR1 and TGFβRIIdomains of the Mab can bind to their respective targets withoutinterfering with each other. As seen in FIG. 54, the binding ofAnti-EGFR1 HC-TGFβRII+TGFβRII-Anti-EGFR1 LC fusion Mab is comparable toanti-EGFR1-TGFβRII, suggesting that there is no interference in bindingto either target due to the construction of the fusion Mab.

9. TGFβRII-Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC (Fmab 12)

Binding ELISAs: Procedure:

The fusion Mab was tested for its ability to bind to its targets inthree different ELISAs: 1) EGFR1 target-binding ELISA, 2) TGFβ-targetbinding ELISA and 3) Bifunctional ELISA.

For the target binding ELISAs, the targets (rhEGFR-Fc chimera or TGFβ)were coated onto NUNC maxisorb plates overnight at 4° C. The plates werewashed and then blocked with superblock at room temperature for 2 hr.Different dilutions of the fusion Mab or the negative control antibodywas added to the plate. The plate was incubated at room temperature for1 hr. Binding of the fusion Mab was detected by the addition of abiotinylated anti-human IgG F(ab)₂ secondary antibody, followed by a 1hr incubation with peroxidase-conjugated streptavidin at roomtemperature. TMB substrate solution was added and the reaction stoppedwith 1N H₂SO₄. The absorbance was measured at 450 nm on a BioTek SynergyH4 Hybrid reader.

For the bifunctional ELISA, rhEGFR-Fc chimera was coated onto NUNCmaxisorb plates overnight at 4° C. The plates were washed and thenblocked with superblock at room temperature for 2 hr. Differentdilutions of the fusion Mab or the negative control antibody was addedto the plate. The plate was incubated at room temperature for 1 hr.After washing, TGFβ was added and the plate was incubated at roomtemperature for 1 hr. The plate was washed and anti-TGFβ-biotin wasadded and the plate incubated at room temperature for 1 hr. The platewas washed and streptavidin-HRP was added and the plate incubated atroom temperature for 1 hr. After washing, TMB substrate solution wasadded and the reaction stopped with 1N H2504. The absorbance wasmeasured at 450 nm on a BioTek Synergy H4 hybrid reader.

Results:

The binding of TGFβRII-Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab tothe target EGFR1 was slightly reduced (FIG. 53) but was higher for TGFβ(FIG. 56) when compared to the binding of anti-EGFR1-TGFβRII. TheTGFβRII-Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab was also testedin a bifunctional ELISA to determine whether the anti-EGFR1 and TGFβRIIdomains of the Mab can bind to their respective targets withoutinterfering with each other. As seen in FIG. 57, the binding ofTGFβRII-Anti-EGFR1 HC+TGFβRII-Anti-EGFR1 LC fusion Mab is reducedcompared to anti-EGFR1-TGFβRII, suggesting that there is interference inbinding to either target due to the construction of the fusion Mab.

REFERENCES

The contents of all references cited herein are incorporated byreference herein for all purposes.

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That which is claimed:
 1. A fusion protein comprising a targeting moietyand an immunomodulating moiety exhibiting bi-specificiity, wherein thetargeting moiety and the immunomodulating moiety are linked by an aminoacid spacer encoded by nucleotide sequence SEQ ID NO: 4, wherein theimmunomodulating moiety is encoded by nucleotide sequence SEQ ID NO: 3or SEQ ID NO: 7, wherein the targeting moiety is an anti-EGFR1 antibodyencoded by nucleotide sequences consisting of SEQ ID NO: 1 and SEQ IDNO: 2, wherein the encoded by nucleotide sequence SEQ ID NO: 3 or SEQ IDNO: 7 is attached via SEQ ID NO: 4 to the C-terminus of SEQ ID NO 1 orSEQ ID NO:
 2. 2. A fusion protein comprising a targeting moiety and animmunomodulating moiety exhibiting bi-specificiity, wherein thetargeting moiety and the immunomodulating moiety are linked by an aminoacid spacer encoded by nucleotide sequence SEQ ID NO: 4, wherein theimmunomodulating moiety is encoded by nucleotide sequence SEQ ID NO: 3or SEQ ID NO: 7, wherein the targeting moiety is an anti-EGFR1 antibodyencoded by nucleotide sequences consisting of SEQ ID NO: 5 and SEQ IDNO: 6, wherein the encoded by nucleotide sequence SEQ ID NO: 3 or SEQ IDNO: 7 is attached via SEQ ID NO: 4 to the C-terminus of SEQ ID NO 5 orSEQ ID NO: 6.